The present invention relates to a novel thermostable DNA polymerase I from Rhodothermus obamensis, which possesses 3xe2x80x2-5xe2x80x2 exonuclease activity and has a preliminary estimated half-life of 35 minutes at 94xc2x0 C., as well as methods for cloning and producing the large fragment of R. obamensis DNA polymerase I, as well as isolated DNA encoding this enzyme and vectors containing the same.
DNA polymerases are important enzymes involved in chromosome replication and repair. These enzymes have also been employed in DNA diagnostics and analysis. In several of these applications, including PCR, thermocycle sequencing, and iso-thermal strand displacement amplification, DNA polymerases must maintain enzymatic activity at temperatures from 50xc2x0 C.-95xc2x0 C. One advantageous source for such polymerases is thermophiles. Here we describe a method for purifying, cloning and expressing Rhodothermus obamensis DNA polymerase I large fragment in E. coli. 
E. coli DNA polymerase I and T4 DNA polymerase were cloned, purified and characterized previously (Joyce C. M. and Derbyshire V. Methods in Enzymology, 262:3-13, (1995); Nossal N. G. et al. Methods in Enzymology, 262: 560-569, (1995)). These enzymes have a variety of uses in recombinant DNA technology including DNA labeling by nick translation, second-strand cDNA synthesis in cDNA cloning, and DNA sequencing.
U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159 disclosed the use of DNA polymerases in a process for amplifying, detecting, and/or cloning nucleic acid sequences. This process, commonly referred to as polymerase chain reaction (PCR), involves the use of a polymerase, primers and nucleotide triphosphates and amplifying existing nucleic acid sequences.
A number of thermostable DNA polymerases have been isolated and cloned from thermophilic eubacteria. The thermostable Bst DNA polymerase from Bacillus stearothermophilus and the Bca DNA polymerase from Bacillus caldotenax have been cloned and expressed in E. coli (Aliotta J. M. et al. Genetic Analysis: Biomol. Engin, 12:185-195, (1996); Uemori, T. et al. J. Biochem. 113:401-410, (1993)). These two DNA polymerases have been used in strand displacement amplification (Milla, M. A. et al. Biotechniques, 24:392-395, (1998)).
DNA polymerases have also been cloned from a number of Thermus species such as T. aquaticus (Lawyer, F. C., et al. J. Biol. Chem. 264:6427-6437 (1989)). T. thermophilus (Asakura, K. et al. J. Ferment. Bioeng., 76:265-269, (1993), and T. filiformis (Jung, S. E. et al. GenBank Accession No. AF030320, (1997)). These characterized Thermus DNA polymerases, belonging to the Family A DNA polymerases, exhibit 5xe2x80x2-3xe2x80x2 exonuclease activity while lacking 3xe2x80x2-5xe2x80x2 proof-reading exonuclease activity. For thermocycling sequencing, a Taq DNA polymerase variant called ThermoSequenase (F667Y) has been constructed that efficiently incorporates dideoxy terminators and dye-terminators (Tabor S. and Richardson C. C.,Proc. Natl. Acad. Sci. USA, 92:6339-6343, (1995); Vander Horn P. B. et al. Biotechniques, 22:758-765, (1996)). Although readable DNA sequence for one sequencing reaction has improved from 300 bp to about 600 bp, further technical improvements are needed to achieve 1000 or more bases of reliable sequence for each reaction. Such improvement most likely requires the introduction of new DNA polymerases such as thermostable T7-like DNA polymerases.
Research was conducted on the isolation and purification of DNA polymerases from Thermus aquaticus (Chien, A. et al. J. Bacteriol. 127:1550-1557, (1976)). The publication of Chien, A. et al. discloses the isolation and purification of a DNA polymerase with a temperature optimum of 80xc2x0 C. from T. aquaticus YT1 strain. The Chien et al., purification procedure involves a four-step process. These steps include preparation of crude extract, DEAE-Sephadex chromatography, phosphocellulose chromatography and chromatography on DNA cellulose.
U.S. Pat. No. 4,889,818 discloses a purified thermostable DNA polymerase from T. aquaticus, Taq DNA polymerase, having a molecular weight of about 86,000 to 90,000 daltons prepared by a process substantially identical to the process of Kaledin with the addition of the substitution of a phosphocellulose chromatography step in lieu of chromatography on single-strand DNA-cellulose. In addition, European Patent Application 0258017 disclose Taq polymerase as the preferred enzyme for use in the PCR process discussed above. Research has indicated that while the Taq DNA polymerase has a 5xe2x80x2-3xe2x80x2 polymerase-dependent exonuclease function, Taq DNA polymerase does not possess a 3xe2x80x2-5xe2x80x2 proofreading exonuclease function (Lawyer, F. C., et al. J. Biol. Chem. 264:6427-6437 (1989)). As a result, Taq DNA polymerase is prone to base incorporation errors, making its use in certain applications undesirable. For example, attempting to clone an amplified gene is problematic since any one copy of the gene may contain an error due to a random misincorporation event. Depending on where in the replication cycle that error occurs (e.g., in an early replication cycle), the entire DNA amplified could contain the erroneously incorporated base, thus, giving rise to a mutated gene product.
Accordingly, it would be desirable to clone and produce a thermostable DNA polymerase with 3xe2x80x2-5xe2x80x2 proof-reading exonuclease activity that may be used to improve the fidelity of DNA amplification reactions described above. It would also be desirable to clone a thermostable and processive DNA polymerase which efficiently incorporates dye terminators.
In accordance with the present invention, there is provided a novel thermostable DNA polymerase I from Rhodothermus obamensis, which possesses 3xe2x80x2-5xe2x80x2 exonuclease activity and has a preliminarily estimated half-life of 35 minutes at 94xc2x0 C. This thermostable enzyme obtainable from Rhodothermus obamensis, a thermophile isolated from a shallow marine hydrothermal vent in Tachibana Bay, Japan, has a molecular weight of about 104 kDa, and possesses a tyrosine residue in the ribosome binding domain which increases the incorporation rate of dideoxynucleotides.
Also provided by the instant invention are methods for cloning and producing the large fragment of R. obamensis DNA polymerase I, as well as isolated DNA encoding this enzyme and vectors containing the same. The Rhodothermus obamensis DNA polymerase I large fragment has a molecular weight of about 71 kDa.